A Concise Approach to Site-Specific Topological ProteinâPoly(amino

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A Concise Approach to Site-Specific Topological Protein-Poly(Amino Acid) Conjugates Enabled by In-situ Generated Functionalities Yingqin Hou, Jingsong Yuan, Yu Zhou, Jin Yu, and Hua Lu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b05413 • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Yingqin Hou,¶ Jingsong Yuan,¶ Yu Zhou, Jin Yu and Hua Lu* Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People’s Republic of China ABSTRACT: Controlling the topology of polymer-modified proteins has attracted growing interest. However, one of the main challenges in this field is the lack of efficient and site-specific methods for installing multiple bioorthogonal functionalities on substrate polymers. We report here an orchestrating strategy that provides easy access to various topological protein-poly(amino acid) (PAA) conjugates in high yields. This method features the in situ installation of two “chemical handles”, including a thioester for native chemical ligation and a polyglycine tag for sortase A-mediated ligation, at both ends of substrate PAAs. As a result, neither pre-functionalization of initiator or monomer units, nor postpolymerization modification of the resultant polymers, is necessary. Site-specific topological conjugates, particularly circular conjugates, can be conveniently synthesized under mild conditions from the functionalized PAAs. The biomedical utility of our method is demonstrated by the rapid and efficient generation of several therapeutic interferon-α conjugates, which exhibited significantly enhanced protease resistance and thermostability. Given the versatility of both PAAs and proteins, the method offers a convenient approach producing libraries of conjugates for biological applications.

INTRODUCTION Protein-polymer conjugation (e.g. PEGylation)1 is widely used to modulate protein functions, effect hierarchical self-assembly and deliver therapeutic agents.2 Recently, controlling the topology of such conjugates has become a nascent field attracting growing attention, inspired by previous findings that cyclized proteins often possess beneficial pharmaceutical and biological properties ranging from enhanced thermal stability to improved protease resistance.3 Despite recent advances,4 a major challenge in protein-polymer conjugates is the generation of heterogeneous populations and poor molecular weight control due to the use of nonselective chemical ligation methods. Moreover, topological conjugates, such as headto-tail circular protein-polymer conjugates, still remain largely unexplored.3c, 3d Often, a key hurdle impeding the synthesis is not the lack of appropriate reactions to ligate the two macromolecules, but the redundant and costineffective process of introducing multiple orthogonal functionalities that can be used for conjugation (e.g. producing heterotelechelic polymers). Therefore, a universal and robust synthetic platform that allows the traceless and precise generation of polymers bearing orthogonal ligation functionalities is highly desired. PEG, albeit effective, is nondegradable, provides very few functionalities, and may leads to accelerated blood clearance (ABC) upon repetitive injections.5 To develop alternative materials of PEG, polypeptides, or poly(amino acid)s (PAA), are promising candidates for their proteinlike backbone, versatile side-chain functionality and

tunable degradability.6 Indeed, the fusion of unstructured polypeptides such as XTEN or elastin-like protein (ELP) to target proteins has been tested, which leads to superior pharmacological properties.7 However, the production of such fusion proteins requires substantial genetic modification, and is usually limited to the 20 canonical amino acids. In comparison, synthetic PAAs generated by the ring-opening polymerization (ROP) of α-amino acid Ncarboxyanhydrides (NCAs)8 are inexpensive, easily accessible in large scale, and can offer expanded chemical diversity and higher-ordered structures exploitable for numerous biomedical applications.9 Despite these advantages, there are only a few reports on randomly labeled protein-PAA conjugates10 and no study has been conducted on their topological control. Herein, we describe a one-pot, two-step polymerization process that produces heterotelechelic block PAAs leading to the facile synthesis of site-specific topological proteinPAA conjugates. Specifically, two orthogonal chemical handles, including a phenyl thioester for native chemical ligation4d,11 (NCL, Figure 1A) and a polyglycine for sortase A mediated ligation12 (SML, Figure 1B), are in situ installed at the C- and N-termini of PAAs, respectively. As a result, neither pre-functionalization of initiator or monomer units, nor post-polymerization modification of the resultant polymers, is necessary. Combinatorial execution of NCL and SML under mild conditions enables the rapid generation of various protein-PAA conjugates with welldefined topological structures, including, in particular, cyclic conjugates (Figure 1C) that exhibit remarkable

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Figure 1. A) Synthesis of poly(amino acid)s (PAAs) by phenyl trimethylsilyl sulfide (PhS-TMS) mediated controlled ROP of NCAs. A thioester was installed to the C-terminus of the PAA and serves as a chemical handle for native chemical ligation (NCL). B) A polyglycine (Gx) tag was generated by the ROP of glycine NCA as a substrate for Sortase A mediated ligation (SML). C) Illustration of various site-specific topological proteinPAA conjugates described in this work. thermostability and protease resistance. Given the abundance and versatility of both PAAs and proteins, the method holds tremendous potential for biological and pharmaceutical applications. RESULTS AND DISCUSSION Synthesis of Protein-PAA NCL Conjugates. We began our study by creating protein-PAA conjugates via NCL. One of the main challenges of this approach lies in the difficulty of constructing a thioester linkage in PAAs, which in fact has been an active field of Fmoc solid phase peptide synthesis (SPPS).13 We have previously demonstrated that phenyl trimethylsilyl sulfide (PhSTMS), a commercially available initiator, could mediate rapid ROP of NCAs and produce well-defined PAAs tethering an in situ generated C-terminal phenyl thioester (PAA-SPh).14 However, the ligation of PAA-SPh to complex substrates, especially macromolecules with rich functionalities, has yet to be established. We therefore sought to first investigate the NCL reactivity of PAA-SPh towards various cysteine-functionalized substrates ranging from small molecules, peptides to proteins. The N-acetylcapped poly(-(2-(2-(2-methoxyethoxy)ethoxy)ethyl Lglutamate) (P(OEG3-Glu)n-SPh, n = feeding M/I ratio, Figure S1-S2), produced by PhS-TMS mediated ROP of (2-(2-(2-methoxyethoxy)ethoxy)ethyl L-glutamate NCA(OEG3 -GluNCA),15 was selected as a model PAA for its excellent aqueous solubility and “stealth” PEG-like side

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Figure 2. Synthesis and characterization of various NCL conjugates. A) NCL of P(OEG3-Glu)n-SPh to N-cysteine functionalized small molecules, peptides and proteins. BC) Fluorescent native gel of P(OEG3-Glu)7-eGFP (B) and P(OEG3-Glu)20-eGFP (C) after purification. chain (Figure 2A). Cys-DUPA (Figure 2A), a smallmolecule ligand binding to the human prostate-specific membrane antigen,16 was ligated to P(OEG3-Glu)7-SPh in a 1:1 molar ratio under room temperature, affording the product with >95% conversion (Figure S3). Similarly, NCL of P(OEG3-Glu)n-SPh (n= 7 or 20) to Cys-PEP, a 18-mer peptide (sequence: CGDAKGLPETGHHHHHHK, MW = 1998 Da; Figure 2A), resulted in complete conversion at 1:13:1 feeding ratios (Figure S4-S5). To generate the Nterminal cysteine necessary for NCL, a short peptide tag ENLYFQC was fused to the N-termini of selected proteins and then cleaved by tobacco etch virus (TEV) protease.17 Utilizing this method, the N-Cys enhanced green fluorescent protein (Cys-eGFP) was successfully produced at a yield of ~48 mg/L. NCL of P(OEG3-Glu)n-SPh (n = 7, 20) and Cys-eGFP at room temperature for 10-12 h led to the efficient formation of well-defined conjugates with conversions ranging from ~76-81% (Figure 2B-C, Figure S6). Importantly, mixing ENLYFQ-Cys-eGFP and P(OEG3Glu)n-SPh under the same condition did not afford any product, indicating the NCL reaction is indeed N-Cys selective. It is also worth noting that our ligation protocol required a significantly lower molar ratio of P(OEG3-Glu)nSPh over Cys-eGFP (3:1 to 5:1) than what would normally be needed by conventional protein-polymer conjugation methods (typically >20:1). Moreover, unlike in standard NCL processes, additives such as 4-mercaptophenylacetic acid were not necessary, which minimized the likelihood of protein destabilization and/or precipitation. Indeed, we did not observe any significant decrease in the


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Figure 3. Synthesis (A) and SDS-PAGE gel characterization (B) of a SML-conjugate eGFP-mPEG. A polyglycine tag was generated by mPEG-NH2 mediated ROP of GlyNCA and served as a substrate of SrtA.

Figure 4. Synthesis and characterization of K-conjugate and D-conjugate. A-B) Synthetic scheme (A) and SDS-PAGE gel analysis (B) of the K-conjugate P(OEG3-Glu)7-eGFP-mPEG. C-D) Synthetic scheme (C) and SDS-PAGE gel analysis (D) of the D-conjugate eGFP-P(OEG3-Glu)7-IFNα. fluorescence of eGFP during the conjugation. Together, these results underscored the high NCL reactivity of PAASPhs towards various substrates, including those challenging and complex biomolecules. Synthesis of Protein-PAA SML Conjugates. We next investigated SML for protein-PAA conjugation owing to its high efficiency and chemoselectivity (SML-conjugate, Figure 1B). Sortase A (SrtA) from Staphylcoccus aureus is an enzyme catalyzing the transpeptidation between an LPXTG motif (X = any canonical amino acid) and the Nterminal amino group of a polyglycine tag. Previously, it has been noted that StrA can accommodate polyglycine substrates of various lengths (Gx, x = 1 to 5),12d which prompted us to test whether the enzyme could also recognize the polyglycine generated by the ROP of glycine NCA (Gly-NCA). To this end, two block copolymers denoted as Gx-mPEG (x = 5 or 10, Figure S7) were prepared

by mPEG-amine (MW = 2000 Da) initiated Gly-NCA polymerization (Figure 3A). After optimizing reaction conditions (Figure S8), SML of Gx-mPEG (5 equiv.) to eGFP bearing a C-terminal LPETGGLEH6 tag (eGFPLPETGGLEH6) proceeded smoothly within 30 min in the presence of 0.1 equiv. of SrtA and afforded the desired products in ~90% conversion (Figure 3B). It should be emphasized that, although Gx is accessible by SPPS or genetic engineering, our method, with comparable SML efficiency,3c,12d is simpler, more scalable, and fully compatible with ROP of NCAs. Synthesis of Protein-PAA K- and D-Conjugates. The combinatorial application of NCL and SML was demonstrated in the synthesis of a wide range of challenging multi-domain18 and circular protein-PAA conjugates. Sequential attachment of P(OEG3-Glu)n-SPh via NCL and Gx-mPEG via SML to Cys-eGFP-LPETGGLEH6



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Figure 5. Synthesis and characterization of C-conjugate circ(P(OEG3-Glu)7-eGFP). A) Synthetic scheme for circ(P(OEG3Glu)7-eGFP). B-D) Successful generation of circ(P(OEG3-Glu)7-eGFP) was confirmed by MALDI-TOF mass spectrum (B), SDS-PAGE gel (C), and anti-H6 tag western blot (D) of circ(P(OEG3-Glu)7-eGFP). E) Degradation kinetics of Cys-eGFP, G3P(OEG3-Glu)7-eGFP-LPETGGLEH6, and circ(P(OEG3-Glu)7-eGFP) incubated with 80 μg/mL carboxypeptidase C; the experiments were repeated as triplicates and error bars were presented as standard deviations.

(Figure 4B and Figure S9) furnished a knot-like polymerprotein-polymer conjugate (K-conjugate, Figure 4A). In another example, a dumbbell-like heterodimeric proteinpolymer-protein conjugate (D-Conjugate) was synthesized from a heterotelechelic block PAA G3-P(OEG3-Glu)7-SPh (Figure S10), which was generated by one-pot, two-step ROP of OEG3-GluNCA and Gly-NCA (Figure 4C) without additional post-polymerization modifications. SML of G3P(OEG3-Glu)7-SPh with eGFP-LPETGGLEH6, followed by NCL of the resultant product with Cys-IFNα, led to the efficient generation of the desired D-Conjugate (Figure 4D and Figure S11). Synthesis of Peptide/Protein-PAA Circular Conjugates.

Our ligation method can also be used to prepare head-totail cyclic protein-PAA conjugates (C-conjugate, Figure 5A), which have remained synthetically inaccessible. In a model reaction, NCL of heterotelechelic G3-P(OEG3-Glu)7SPh and bifunctional Cys-PEP (sequence: CGDAKGLPETGHHHHHHK) was conducted to afford the linear intermediate, which then readily underwent SMLmediated cyclization to generate the desired C-conjugate in 61% overall yield. The cyclization product was unambiguously confirmed by MALDI-TOF mass spectrometry (Figure S12). Next, we used Cys-eGFPLPETGGLEH6 as the cyclization partner with G3-P(OEG3Glu)7-SPh. Again, the two substrates were first ligated via NCL into the linear intermediate and then cyclized by SML in a diluted solution (~ 1.0 - 1.5 mg/mL). This conjugation sequence resulted in the cleavage of a GGLEH6 tag (1160 Da) from the cyclization product, which allowed the reaction to be readily monitored by both MALDI-TOF mass spectrometry and Western blotting, and enabled convenient purification by passing the resultant mixture

through a NiNTA column and collecting the flow-through. Indeed, the formation of the C-conjugate circ(P(OEG3Glu)7-eGFP) was confirmed by MALDI-TOF analysis that indicated a MW decrease of ~1200 Da (Figure 5B), a shift of the eGFP protein band toward the low MW range (Figure 5C), and the disappearance of the C-conjugate band in the anti-H6 western blot (Figure 5D). In contrast, P(OEG3-Glu)7-eGFP-LPETGGLEH6, which contained no Nterminal polyglycine, did not cyclize in the presence of StrA under similar conditions. A ~42% purified yield was obtained for circ(P(OEG3-Glu)7-eGFP) with ~95% purity and less than 5% higher-MW byproduct (Figure S13B-D). In all cases, the SML method that we developed required only 0.05-0.1 equiv. of SrtA and 30 min of reaction time, whereas previously published SrtA-based protein/peptide cyclization examples were considerably less efficient in comparison (typically 0.33-1 equiv. of SrtA and 16-88 h of reaction time).3c,19 Moreover, no proteolytic degradation of circ(P(OEG3-Glu)7-eGFP) was observed up to 7 h when it was incubated with carboxypeptidase C, a C-terminal protease with broad amino acid specificity. This result offered additional evidence for cyclization, which was consistent with the unavailability of the C-terminal carboxylic acid due to the successful cyclization. In contrast, when subjected to the same treatment, ~80% of the linear precursor G3-P(OEG3-Glu)7-eGFP-LPETGGLEH6 and almost 100% of Cys-eGFP were degraded in ~3 h and ~1.5 h, respectively (Figure 5E and Figure S14A-C). Notably, a higher substrate concentration (e.g. 6.0 mg/mL) led to a significantly lower cyclization yield (~13%) due to precipitation of protein in SML buffer. To examine the efficiency of PAAs with greater MWs, we subjected G3P(OEG3-Glu)30-SPh (MW ~8500 Da) to protein cyclization


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Figure 6. Protease resistance and thermostability of wt and polymer-conjugated IFNα. A) Cartoon illustration of IFNα conjugates prepared in this study. B) Degradation kinetics of wt-IFNα, G3-P(OEG3-Glu)7-IFNα and circ(P(OEG3-Glu)7-IFNα) by trypsin (0.5 μg/mL). C) Melting temperatures (Tm) of wt-IFNα, IFNα-mPEG, G3-P(OEG3-Glu)7-IFNα and circ(P(OEG3Glu)n-IFNα) ( n =7 and 20) measured by thermofluor assay. D) CD spectra of wt-IFNα and circ(P(OEG3-Glu)7-IFNα) at 37 oC and 65 oC. E) Degradation kinetics of wt-IFNα, circ(P(OEG -Glu) -IFNα) and circ(P(OEG -Glu) -IFNα) by trypsin(2.5 3 7 3 20 μg/mL). All experiments were repeated as triplicates and error bars were presented as standard deviations. under SML condition. The cyclized product was obtained at a ~15% yield after purification (data not shown). Protease Resistance and Thermostability of Various

Interferon-α PAA Conjugates. Wild-type interferon-α20 (wt-IFNα) is an important therapeutic protein that often requires polymer modification in clinical applications due to its suboptimal in vitro and in vivo stability. To demonstrate the benefits of PAA modification, we prepared several IFNα conjugates with ~62-80% purified yields, including a NCL conjugate G3-P(OEG3-Glu)7-IFNα, a SML conjugate IFNα-mPEG, and a C-conjugates circ(P(OEG3-Glu)7-IFNα), using the ligation protocol described earlier (Figure 6A and Figure S15-S17). All three substrate polymers used in conjugation have nearidentical molecular weights (~2000 Da) to minimize the impact of mass differences. Trypsin digestion assay indicated that both circ(P(OEG3-Glu)7-IFNα) and G3P(OEG3-Glu)7-IFNα were significantly more proteaseresistant than IFNα-PEG and wt-IFNα (Figure 6B and Figure S18-19), which were rapidly degraded. Next, we examined the thermal stability of the free and conjugated IFNαs by measuring their melting temperatures (Tm) in a thermofluor assay (Figure 6C and Figure S20).3c Interestingly, both IFNα-PEG (49.2 oC) and G3-P(OEG3Glu)7-IFNα (54.6 oC) showed lower Tm’s than wt-IFNα (59.0 oC); in contrast, the T m of circ(P(OEG3-Glu)7-IFNα

increased to 62.6 oC, which is comparable to the reported value of another cyclized IFNα.3c To further verify circ(P(OEG3-Glu)7-IFNα)’s enhanced thermostability, the conjugate and wt-IFNα were separately subjected to a heating ramp from 37 to 65 oC over a course of 90 min. Circular dichroism (CD) spectroscopy detected no obvious loss of helicity in circ(P(OEG3-Glu)7-IFNα) after the heat shock, whereas wt-IFNα became denatured and precipitated at ~65 oC (Figure 6D). Similarly, circ(G3P(OEG3-Glu)7-IFNα) undergoing lyophilization showed no change in its CD pattern, suggesting its ability to withstand freeze-drying without denaturation (Figure S21). It should be emphasized that our circ(P(OEG3-Glu)7-IFNα) showed comparable in vitro anti-proliferation activity to wt-IFNα in Daudi cells, indicating that the cyclization did not affect their biological activities (Figure S22). Together, these results confirmed that a circular topology can protect protein conjugates against various environmental stresses including protease degradation and heat shock. To examine how the MW of PAAs affects the protease and thermal stability of C-conjugates, we synthesized the much bulkier circ(P(OEG3-Glu)20-IFNα) and incubated it with trypsin at a concentration 5 times higher than what was used in Figure 6B. Compared to the smaller circ(P(OEG3-Glu)7-IFNα), which showed slightly enhanced resistance over wt-IFNα under this condition, almost no degradation of circ(P(OEG3-Glu)20-IFNα) was detected up



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to 2 h of trypsin treatment (Figure 6E and Figure S23). In thermofluor assay, circ(P(OEG3-Glu)20-IFNα) showed a Tm value of 70.1 oC, which was ~ 11.1 oC higher than that of wtIFNα (Figure 6C and Figure S20) and arguably the highest value among all reported IFNα variants so far. Overall, these results indicated that C-conjugates with higher MWs tend to be more protease-resistant and thermostable. CONCLUSIONS In conclusion, we developed a concise and orchestrating strategy for the one-pot production of heterotelechelic PAAs, which led to rapid and efficient generation of well-defined topological protein-PAA conjugates. Particularly, the current study provided the first synthesis of head-to-tail circular protein-PAA conjugates to our best knowledge and demonstrated the effectiveness of cyclization on improving the thermostability and protease resistance of proteins such as eGFP and IFNα. We envision that our conjugation method would enable convenient access to a wide range of welldefined protein-PAA conjugates for numerous biological and medical applications. We are currently investigating the in vivo anticancer efficacies of various therapeutic protein conjugates including IFNα.

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Supporting Information. Materials and Methods, instrumentations, 1H NMR, UPLC-ESIMS, MALDI-TOF MS, and SDS-PAGE data. This material is available free of charge via the Internet at http://pubs.acs.org.

*E-mail: [email protected] (5) ¶ Y.H

and J.Y. contributed equally to this work. (6)

This work is supported by grants from National Natural Science Foundation of China (NSFC21474004 and NSFC21434008) and State High-Tech Development Program of China (863 Program No. 2015AA020941). H.L. thanks the support from the Youth ThousandTalents Program of China. We thank the mass spectrometry facility of National Center for Protein Sciences at Peking University and Dr. Wen Zhou for assistance with MALDI-TOF analysis.

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The authors declare no competing financial interest.

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